Summary/Abstract Blood vessels play an important role in the regulation of arterial blood pressure (BP). Precise BP regulation requires coordination between vasodilator and vasoconstrictor signals in the endothelium (EC) and smooth muscle (SMC). EC-derived nitric oxide (NO) is among the key signals which instruct the SMC to dilate or contract. Our studies show that the NO pathway is coordinately regulated through transcriptional and post- translational pathways initiated by PPAR?, a nuclear receptor transcription factor. Our data support the concepts that PPAR?: 1) acts as a sensor in EC to regulate redox state, and through this, bioavailability of NO, and 2) regulates the responsiveness of SMC to NO by independently controlling a) a RhoA/Rho kinase (ROCK) activity that promotes constriction, and b) production and stability of cyclic GMP (cGMP), a critical mediator of vasodilation. The range of PPAR?-dependent molecular mechanisms in both cell types is surprisingly complex; requiring novel transcriptional co-factors (e.g. retinol binding protein 7; RBP7) which form a transcriptional regulatory hub with PPAR?, and post-translational regulation of critical SMC mediators (RhoA and phosphodiesterase 5, PDE5) by Cullin-3 E3 ubiquitin ligase-mediated protein turnover. Importantly, this PPAR? initiated ?final common pathway? has profound effects on vasomotor function, BP and vascular stiffness, and the studies proposed herein have potential implications for the treatment of these disorders. However, the signals which initiate and mediate these responses and the range of molecular targets remain poorly understood. This proposal will focus on two distinct PPAR?-regulated pathways. We will examine the PPAR?-RhoBTB1-Cullin-3 pathway in smooth muscle and will 1) determine if the RhoBTB1-Cullin-3 pathway can be exploited as a potential future therapeutic by assessing if RhoBTB1 can protect and reverse phenotypes in models of hypertension or in other disease models in which vascular dysfunction is a comorbidity, 2) determine if RhoBTB1 is important in other cells types including endothelium, and 3) employ a proteomic strategy to identify novel RhoBTB1 binding partners and Cullin-3 substrates in vascular smooth muscle. We will also examine the PPAR?-RBP7-anti-oxidant pathway in endothelium and will perform 1) structure function analysis to identify key mechanisms regulating PPAR? transcriptional activity by RBP7, and 2) genome wide transcriptome and chromatin immunoprecipitation studies to assess the contribution of RBP7 to mediated transcriptional activity of PPAR? and its obligate heterodimer RXR. This program will lead to new concepts and directions of investigation for the field and will not only deepen our understanding of the role of two fundamentally important pathways in the vasculature, but will also address fundamental transcriptional and post-translational mechanisms that are of relevance in many cell types.